Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

Comparison of five methods for oxacillin susceptibility testing of Staphylococcus aureus isolates from cystic fibrosis patients

Methicillin-resistant Staphylococcus aureus (MRSA) are now a worldwide problem. Cystic fibrosis (CF) patients are commonly colonized and infected by MRSA. Accurate oxacillin susceptibility testing is mandatory for the adequate management of these patients. We performed a comparison of the accuracy of different tests in CF isolates, including methicillin-susceptible S. aureus and MRSA with different SCCmec types, and using the mecA gene as the gold-standard. The sensitivity and specificity of oxacillin disc, Etest, and oxacillin agar screening plate were 100%. Sensitivity of the cefoxitin disc was 85% and specificity was 100%. For clinically relevant isolates, laboratories may consider the use of a combination of two phenotypic methods.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil

INTRODUCTION: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.

Isolation of Pseudomonas cepacia in cystic fibrosis patient

Pulmonary infection on cystic fibrosis (CF) patients are associated with a limited qualitative number of microorganisms. During the colonization process, Staphylococcus aureus usually preceedes Pseudomonas aeruginosa. This latter is at first non-mucoid, being replaced or associated to a mucoid morphotype which is rare in other diseases. In 1980, Pseudomonas cepacia appeared as an important agent in CF pulmonary infections with a mean frequency of about 6.1% isolations in different parts of the world. The primus colonization mainly occurs in the presence of pre-existent tissue lesions and the clinical progress of the disease is variable. In some patients it can be fulminant; in others it can cause a gradual and slow decrease in their pulmonary functions. The concern with this germ isolation is justified by its antibiotic multiple resistence and the possibility of direct transmission from a colonized patient to a non-colonized one. We reported the first case of P. cepacia infection in a CF patient in our area. The microbiological attendance to this patient had been made from 1986 to 1991 and the first positive culture appeared in 1988. The sensitivity profile showed that the primus colonization strain was sensitive to 9 of 17 tested antibiotics, however in the last culture the strain was resistent to all antibiotics. These data corroborate the need for monitoring the bacterial flora on CF patients respiratory system.

Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

Structure and function of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Effect of meconium ileus on the clinical prognosis of patients with cystic fibrosis

The objective of the present study was to determine the possible prognostic factors which may explain the difference in the survival of patients with cystic fibrosis (CF) with and without meconium ileus. Over a period of 20 years, 127 patients with CF, whose diagnosis was confirmed by typical clinical characteristics and altered sweat chloride levels, were studied retrospectively. The patients were divided into two groups: group 1 consisted of patients who presented CF and meconium ileus (N = 9), and group 2 consisted of patients with CF without meconium ileus (N = 118). The characteristics studied were based on data obtained upon admission of the patients using a specific protocol. Demographic, clinical, nutritional and laboratory data were obtained. The genotype was determined in 106 patients by PCR. Survival was analyzed using the Kaplan-Meier method. The median follow-up period was 44 months. A statistically significant difference was observed between the groups studied regarding the following variables: age at diagnosis and weight and height z scores. The presence of meconium ileus was associated with an earlier diagnosis; these patients had greater deficits in height and weight at the time of diagnosis and at the end of the study. The estimated probability of survival for patients with CF without meconium ileus was 62 ± 14% and for those with meconium ileus 32 ± 18%. Patients with CF and meconium ileus presented a poor nutritional status at diagnosis and a lower survival rate compared to the general CF population.

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7%) of 230 unrelated CF alleles. Fifteen (13.0%) patients were homozygous for this mutation, while 20 (17.4%) were heterozygous; the remaining 80 (69.6%) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2%) patients, 21 (18.3%) had the sequence variation 4521G/A, 11 (9.6%) had a not yet described sequence variation 4407T/A and 8 (7.0%) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

Cystic fibrosis with normal sweat chloride concentration: case report

Cystic fibrosis is a genetic disease usually diagnosed by abnormal sweat testing. We report a case of an 18-year-old female with bronchiectasis, chronic P. aeruginosa infection, and normal sweat chloride concentrations who experienced rapid decrease of lung function and clinical deterioration despite treatment. Given the high suspicion ofcystic fibrosis, broad genotyping testing was performed, showing a compound heterozygous with deltaF508 and 3849+10kb C->T mutations, therefore confirming cystic fibrosis diagnosis. Although the sweat chloride test remains the gold standard for the diagnosis of cystic fibrosis, alternative diagnostic tests such as genotyping and electrophysiologic measurements must be performed if there is suspicion of cystic fibrosis, despite normal or borderline sweat chloride levels.

Use of PCR for the determination of the frequency of the deltaF508 mutation in Brasilian cistic fibrosis patients

The [Delta]F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion

Adaptação cultural e propriedades psicométricas iniciais do instrumento DISABKIDS ® – Cystic Fibrosis Module – versão brasileira

Este estudo objetivou adaptar culturalmente e descrever as propriedades psicométricas iniciais do instrumento de mensuração de qualidade de vida relacionada à saúde DISABKIDS® − Cystic Fibrosis Module para crianças e adolescentes e seus pais/cuidadores. Estudo metodológico de desenvolvimento sequencial, incluindo 126 participantes em quatro estados brasileiros. Envolveu tradução e retrotradução dos itens, equivalência conceitual e semântica, validade de face e descrição das propriedades psicométricas iniciais relacionadas ao construto e fidedignidade . Para equivalência semântica da versão adaptada houve ajustes na redação de um item. O instrumento apresentou consistência interna satisfatória com valores de alfa de Cronbach entre 0,70 e 0,85, validade convergente com valores de correlação acima 0,40 em 85% dos itens e validade divergente com valores de ajuste superiores a 75%. A versão brasileira do DISABKIDS® - CFM certamente se constituirá em um instrumento válido e confiável para a mensuração da qualidade de vida de crianças e adolescentes brasileiros com fibrose cística.

Analysis of the DF508 mutation in a Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients

Sixty-one cystic fibrosis patients admitted for check-up or antibiotic treatment were enrolled for genetic and clinical evaluation. Genetic analysis was performed on blood samples stored on neonatal screening cards using PCR techniques to determine the presence of DF508 mutations. Clinical evaluation included Shwachman and Chrispin-Norman scores, age at onset of symptoms and diagnosis, spirometry, awake and sleep pulse oximetry, hyponychial angle measurement and presence of chronic Pseudomonas aeruginosa colonization. Eighteen patients (29.5%) were homozygous for the DF508 mutation, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, corresponding to a 50.8% prevalence of the mutation in the whole population. Analysis by the Kruskal-Wallis test for comparison of genetic status with continuous variables or by the chi-square test and logistic regression for dichotomous variables showed no significant differences between any two groups for a = 0.05. We conclude that genetic status in relation to the DF508 mutation is not associated with pulmonary status as evaluated by the above variables

Neonatal screening for cystic fibrosis in São Paulo State, Brazil: a pilot study

Cystic fibrosis is one of the most common autosomal recessive hereditary diseases in the Caucasian population, with an incidence of 1:2000 to 1:3500 liveborns. More than 1000 mutations have been described with the most common being F508del. It has a prevalence of 23-55% within the Brazilian population. The lack of population-based studies evaluating the incidence of cystic fibrosis in São Paulo State, Brazil, and an analysis concerning the costs of implantation of a screening program motivated the present study. A total of 60,000 dried blood samples from Guthrie cards obtained from April 2005 to January 2006 for neonatal screening at 4 reference centers in São Paulo State were analyzed. The immunoreactive trypsinogen (IRT)/IRT protocol was used with the cut-off value being 70 ng/mL. A total of 532 children (0.9%) showed IRT >70 ng/mL and a 2nd sample was collected from 418 (80.3%) of these patients. Four affected children were detected at two centers, corresponding to an incidence of 1:8403. The average age at diagnosis was 69 days, and 3 of the children already showed severe symptoms of the disease. The rate of false-positive results was 95.2% and the positive predictive value for the test was 8%. The cost of detecting an affected subject was approximately US$8,000.00 when this cystic fibrosis program was added to an existing neonatal screening program. The present study clearly shows the difficulties involved in cystic fibrosis screening using the IRT/IRT protocol, particularly in a population with no long-term tradition of neonatal screening.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.